Controllable Preparation and Performance Investigation of Amino Acid Functionalized Mixed-Mode Chromatographic Stationary Phase

Amino acids with hydrophilic, hydrophobic and ion-exchange sites offer great potential for the design of mixed-mode chromatographic stationary phases. This study developed controllable strategies for preparing two types of amino acid-functionalized stationary phases via peptide grafting and hyperbranched modification using polystyrene-divinylbenzene-glycidyl methacrylate (PS-GMA) as the substrate. l-Alanine (Ala), l-lysine (Lys), and l-tyrosine (Tyr) were immobilized via dry-wet cycle peptide formation or 1,4-butanediol diglycidyl ether cross-linking. Structural characterization confirmed successful grafting, and chromatographic evaluations revealed mixed-mode retention behavior for aromatic compounds and nucleosides. The Lys based materials exhibited superior performance, attributable to their ability to facilitate diverse intermolecular interactions. The peptide grafting approach markedly reduced mass transfer resistance, thereby improving separation efficiency and theoretical plate numbers. Meanwhile, the hyperbranched modification method constructed a three-dimensional architecture on the surface, further enhancing spatial selectivity. Notably, the hyperbranched Lys stationary phase (Lys-4L-HBSP) achieved high accuracy (recoveries: 99.96–108.41%) and excellent linearity (R2 > 0.99) in the determination of nitrophenol isomer pollutants in spiked river water samples. This work highlights the role of amino acid selection and grafting strategy in tuning chromatographic performance, offering a design platform for complex sample separations.